Configuration of glycosaminoglycans

ABSTRACT

The present invention provides stable glycosaminoglycan (GSG) structures and methods of use of such GAG structures. These structures comprise a core of free GAG, a coating of crosslinked GAG surrounding the core, and a layer of a positively charged moiety surrounding the crosslinked GAG layer. These GAG structures provide improved stability, both in in vivo and external use. Furthermore, resurfacing of the structures provides improved cell adhesion and thus improved delivery of the GAG into living cells and tissues.

This Application is a Divisional Application and claim a Priority Dateof Aug. 1, 2003 benefited from a patent application Ser. No. 10/632,599filed by the same inventor of this Patent Application. Patentapplication Ser. No. 10/632,599 is a Formal Application and claim aPriority Date of Aug. 1, 2002 benefited from a Provisional PatentApplication 60/400,078 filed by the same inventor of this PatentApplication.

FIELD OF THE INVENTION

The present invention relates generally to structures composed ofglycosaminoglycans with or without living cells, methods of producingglycosaminoglycan structures, and methods of treating subjects usingthese structures.

BACKGROUND OF THE INVENTION

Slow healing or lack of healing of dermal wounds (e.g., decubitusulcers, severe burns and diabetic ulcers) and eye lesions (e.g., dry eyeand corneal ulcers), is a serious medical problem, affecting millions ofindividuals and causing severe pain or death in many patients. Healingof surgical wounds can also be slow or otherwise problematic,particularly in aging and diabetic individuals. Although wounds may bequite dissimilar in terms of cause, morphology and tissue affected, theyshare a common healing mechanism. Each repair process ultimatelyrequires that the correct type of cell migrate into the wound insufficient numbers to have an effect: macrophages to debride wounds,fibroblasts for the formation of new collagen and other extracellularmatrix (ECM) components in wounds where the extracellular matrix wasdamaged, capillary endothelial cells to provide the blood supply, andepithelial cells to ultimately cover the wounds.

However, under certain circumstances, such as burn wounds, and heretolacking of sufficient living skin to support the regeneration of thewound, and then the wounds will last longer and have chances to developsevere infection that some time causes the loss of the lives. Henceresulted hypertrophic burn scars are notoriously difficult to treatbecause of their extensive tissue involvement.

The standard method for grafting extensive or deep burn wounds usedfull-thickness sheet grafts or narrowly meshed, thick, split-thicknessskin grafts [Lattari, et al. J Burn Care Rehabil 18:147-155 (1997)].This method, however, creates an additional complication-prone wound atthe donor site. Donor sites can be painful and may develop infection,hypertrophic scarring, blistering, and hyper- or hypopigmentation. Theproblem of donor site scar hapertrophy occurs most frequently when agraft is taken at more than 0.012 inch thick, leaving a residual dermalbed that is too thin. Meanwhile, early and permanent coverage ofextensive burn wounds is still difficult because of the shortage of thedonor sites.

The unwounded dermis owes much of its structure and strength to theinteractions of cells with the ECM. It is well understood now thatmigration of fibroblasts and keratinocytes plays an important role inwound healing. The ECM is the key dynamic assemblage of interactingmolecules that regulate cell functions and interactions in response tostimulation of wounds. This matrix includes several proteins known tosupport the attachment of a wide variety of cells, includingfibronectin, vitronectin, thrombospondin, collagens, and laminin.Although fibronectin is found at relatively low concentrations inunwounded skin, plasma fibronectin deposition occurs soon afterwounding. When tissue is damaged, the ECM must be replaced to provide ascaffold to support cell attachment and migration. In addition toproviding a scaffold, extracellular matrices can also direct cellularproliferation and differentiation. An extracellular matrix can,therefore, direct healing of a tissue in such a way that the correcttissue geometry is restored.

Acceleration of the healing process can be greatly aided by a betterunderstanding of the factors that influence the synthesis of granulationtissue, which fills the wound before epithelialization. An importantphase of early wound healing involves fibroblast secretion ofglycosaminoglycans (GAGs), which form a hydrophilic matrix suitable forremodeling during healing. Tissue-engineering techniques generally focuson mimicking the ECM by creating a scaffolding of resorbable materialsthat serves to promote wound healing. However, the use of GAGs in suchmaterials is hindered by the instability of free GAGs.

Modification of GAGs, such as hyaluronan, in order to provide morestable structures has been an area of interest in this field. Forexample, U.S. Pat. No. 4,851,521 describes esters of hyaluronic acid inwhich all or only a portion of the carboxylic groups of the acid areesterified. See also Kuo J W et al., Bioconjugate Chem 2:232-241 (1991).These GAG modifications however, alter the biological activity of theGAGs and renders them less effective than their free counterparts. Inaddition, structures formed with these esterified GAGs are instable whenin contact with liquid, such as body fluids, and thus structurescomposed of these molecules do not retain their integrity followingapplication to a subject.

There is a need in the art for compositions, devices and methods forproviding site-specific GAG administration, and in particular for GAGstructures that are stable and cell or tissue accessible in vivo andwhich provide bioavailable GAG.

SUMMARY OF THE INVENTION

The present invention provides stable glycosaminoglycan (GSG) structuresand methods of use of such GAG structures. These structures comprise acore of free GAG, a coating of crosslinked GAG surrounding the core, anda layer of a positively charged moiety surrounding the crosslinked GAGlayer. These GAG structures provide improved stability, both in in vivoan external use. Furthermore, resurfacing of the structures providesimproved cell adhesion and thus improved delivery of the GAG into cells,tissues and/or organs.

In one aspect of the invention, methods are provided for preparingcompounds having of a core of free GAG, a surrounding layer ofcross-linked GAG, and a layer of a positively charged moiety. The methodcomprises the steps of cross-linking an outer layer of a GAG substratesolution (premade into a structure of any shape) with a highconcentration (e.g., between about 35% to 85%) of a cross-linker, whicheffectively crosslinks the GAGs on the periphery of the substrate whileleaving the GAGs in the core of the substrate free, ie., notcrosslinked. The GAG structures can be formed into a desired shape priorto cross-linking, and the crosslinkers help to fix and stabilize thestructures in the particular shape. Preferably, the cross-linkingreagent is an aldehyde, e.g., glutaraldehyde, formaldehyde and the like.GAG structures formed according to the invention, are particularlyadvantageous in that they do not dissolve upon contact with bodilyfluids and provide cells access to bioavailable, free GAG. Also, as anyexcess crosslinking agent can be removed from the structures withoutaffecting the structural integrity, these structures are non-toxic uponadministration.

A particular embodiment of the invention provides a compositioncomprising the GAG structures of the invention and an appropriateexcipient. The excipient may be any acceptable carrier of the GAGstructures of the invention, including water.

It is thus an object of the present invention to provide a polymericcomposition that can provide for directed release of GAGs and can serveas a substrate for cell growth.

In a particular embodiment of the present invention, the GAG structuresare used in or on devices and/or compositions that promote woundhealing.” The GAG structure can provide bioactive GAG in a controlledmanner, e.g., by application externally to a dermal wound, or internallyto a damaged organ or an incised vein or artery with or without livingcells. The GAG structure can be cultured with various cells prior touse, e.g., hyaluronan (HyA) structures can be cultured withkeratinocytes or fibroblasts for use in a liquid bandage.

In one aspect of the invention, the GAG is introduced to a wound siteusing a delivery vehicle such as a solid wound dressing, a liquidbandage, an adhesive and the like, which delivery vehicles comprises theGAG structures of the invention.

It is thus an object of the present invention to provide drug deliverydevices, particularly wound dressings, comprising such polymericdelivery vehicles for release of wound-healing agents to aid in thewound healing process.

In another embodiment of the invention, the GAG structures are providedto modulate other GAG-mediated events, e.g., resurfaced GAG structuresmay be introduced into the synovial fluid of a joint to alleviatearthritis.

It is thus an object of the present invention to provide methods oftreatment for GAG-mediated physiological conditions and/or disease.

An advantage of the present invention is that the cross-linking agent isremoved from the delivery device, thus limiting potential toxicity fromthe cross-liking agents.

It is another advantage of the present invention that the GAG structuresare stable in vivo for up to 6 months.

It is yet another advantage that the GAG structures of the inventionprovide enhanced cell attachment. These and other objects of theinvention will be apparent from the following description and appendedclaims, and from practice of the invention.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1 is a graph illustrating the structural stability of HyAstructures crosslinked with 25% (circles) and 50% (triangles)concentrations of glutaraldehyde for different time periods.

Table 2 is a graph illustrating the wound closure time for theexperimental rats with HyA treated wounds and control groups that thewounds were not treated by HyA grafts but a saline wash.

Table 3 is a graph indicating the comparison of the scar areas of thedifferent groups. The data were collected by the measurement of theexact area on the tissue'histologic stained slides by Confocolmicroscopy.

FIG. 1 is a figure of the modified HyA strand that shows the natural HyAcore with a modified outside layer.

FIG. 2 is a photograph demonstrating that the fabricated HyA strand istransparent, soft and conformable to fit into many kinds of wounds.

FIGS. 3A, 3B, 3C, and 3D are photographs indicating the function of thesurface modification by polylysine. A. Cells are sliding away from theHyA strands without polylysine treatment. B. After the surface has beentreated by polylysine, The HyA strand becomes not cell repellant. C.Fibroblasts are growing on the HyA strand. D. PCNA positively stainingshows that the cell's proliferation is enhanced.

FIGS. 4A, 4B, and 4C are photographs showing an intact stainedresurfaced HyA structure. A. The photograph showing a stained resurfacedHyA intact structure, B. In this HyA strand, the outer core has beensmashed to reveal the inner core of free HyA, C. A photographillustrating the ability of fibroblasts to access the core of free HyAin a resurfaced HyA structure

FIGS. 5A, 5B, 5C, and 5D are photographs illustrating HyA graftimplantation into the rat. A. Full thickness incisions were made on therat's dorsal upper back. Left side wound was treated with HyA strands,right side with only saline washed. B. Three days after the surgery. HyAstrand treated wound healed without contraction. While the right sidewound was still open, bled and contracted. C. Five days post-surgery.The left wound healed with a clear fine line. The right side woundhealed with blood clot and contraction. D. Ten days after surgery. Theleft side wound fully healed with merely visible line. The control side(right) wound showed a typical contracture as a usual healing result.

FIGS. 6A and 6B are photographs indicating the histologic morphology ofthe ten day's wound recovery area. A: the left side wound treated withmodified HyA graft healed with less scar and few inflammatory cellsinfiltration. B. the right side control wound without the treatment hada much large area of scar with fibroblasts proliferation andinflammatory cells infiltration.

DETAILED DESCRIPTION OF INVENTION

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular methods andcompositions described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either-or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aglycosaminoglycan” includes a plurality of such glycosaminoglycanmolecules and reference to “the cross-linking agent” includes referenceto one or more cross-linking agent and equivalents thereof known tothose skilled in the art, and so forth. The publications discussedherein are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

DEFINITIONS

The terms “glycosaminoglycan” and “GAG”, as used interchangeably herein,refer to a macromolecule comprised of carbohydrate. The GAGs for use inthe present invention may vary in size and be either sulfated ornon-sulfated. The GAGs which may be used in the methods of the inventioninclude, but are not limited to, hyaluronic acid, chondroitin sulfates(e.g., neurocan and brevican), laminin, keratin sulfate, chitin andheparin. The term as used herein is intended to encompass modified GAGs(e.g., sulphated GAGs, and GAGs complex with heavy metal ions such asCu2+ and Zn2+) as well as naturally occurring forms.

The terms “free glycosaminoglycan” and “free GAG” as usedinterchangeably herein refer to GAGs that are not chemicallycross-linked or substantially chemically cross-linked. Free GAGs arepreferably available in a naturally occurring form, including but notlimited to a monomer or a dimer. The term is also intended to encompassGAGs that are modified to be in a more active form and accessible to theappropriate cells. As used herein, this term generally describes thebioavailable GAG molecules present in the core of the GAG structures.The core of the GAG structures of the invention comprise at least 50%free GAG, more preferably about 75% free GAG, and even more preferablyat least 90% free GAG. Specific terms such as “free HyA” and “freeheparin” refer to a free form of that particular GAG.

The term “glycosaminoglycan structure”, “GAG structure”, “cross-linkedGAG structure” and the like as used herein refer to a structure havingan external coating of crosslinked GAG and an internal core of free GAG.These structures may or may not have an additional coating of apositively charged molecule, e.g., polylysine. Specific terms such as“HyA structure” refer to a GAG structure composed of the particular GAG.The “GAG structure” may optionally be resurfaced.

The term “glycosaminoglycan substrate”, “GAG substrate” and the like asused interchangeably herein refers to a free glycosaminoglycanpreparation that is exposed to the cross linking agent to produce theglycosaminoglycan structure. The GAG substrate may be a driedpreparation of GAG, or it may be in a liquid form upon exposure to thecross linking agent. The “GAG substrate” may optionally be resurfaced.

The term “resurfaced GAG structure” as used herein refers to a GAGstructure with an external coating of a charged molecule. Preferably thecharged molecule is a suitable polymer with constituent primary aminegroups, including polyvinylamines, polyacrylamides, and polyamino acids,such as polyaspartic acid, polyglutamic acid, and preferably polylysine.

The term “isolated” means the substance of interest is removed from itsnatural surroundings. However, some of the components found with it maycontinue to be with an “isolated” protein. Thus, an “isolated GAG” isnot as it appears in nature but may be substantially less than 100% pureGAG (for example, but not limited to, 50%, 85%, or 90-95% pure.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for disease and/oradverse effect attributable to the disease. In one embodiment,“treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the conditionfrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it; (b) inhibiting the condition,i.e., arresting its development; or (c) relieving the condition, i.e.,causing regression of the disease. The present invention is especiallypertinent to the treatment of a wound, for which the GAG treatment canbe used to facilitate healing. The present invention also pertains totreatments in which delivery of a GAG to a specific site will treat adisease, such as delivery of GAG to synovial fluid to treat arthritis.

GENERAL ASPECTS OF THE INVENTION

The present invention is based in part on the surprising finding thatuse of a high concentration of a cross-linking agent, such asglutaraldehyde, resulted in the cross linking of the GAG molecules onthe edge of the treated structure, but not in the core. This results ina structure having an outer coating of crosslinked GAG, and an innercore of free, bioactive GAG. The invention provides a composition thathas the ability to maintain its structural integrity upon contact with aliquid (e.g., a body fluid) due to the cross-linked outer coating, andyet maintain the bioavailability of the GAG by providing free GAG in thecore. The free GAG in the cores of such structures is accessible tocells exposed to these structures, as the outer cross-linked coatingdoes not impede the ability of a cell (e.g., a fibroblast) to sendoutgrowths into the free core. In addition, by resurfacing thestructure, e.g., by adding a charged outer coating to the GAG structure,providing for better adherence of cells to the GAG structures and fordelivery of free GAG to cells.

Central to the present invention is the high concentration of thecross-linking agent. The cross-linking agent must be of a sufficientconcentration to allow cross-linkage of the outer layer of the GAGwithout substantially crosslinking the internal GAG. Preferably thecrosslinking agent is provided in at least a 35% to 99% solution, morepreferably in a 40% to 85% solution, and even more preferably in a50%-75% solution. The cross-linker used to prepare the structures of theinvention can be any agent with the ability to cross-link either asingle GAG to itself, e.g., HyA-HyA crosslinking, or to crosslinkdifferent GAGs to one another, e.g., HyA-Heparin crosslinking. Exemplarycross-linking agents that may be used in the methods of the inventioninclude, but are not limited to: glutaraldehyde, formaldehyde, vinylsulphone, biscarbodiimides, carbodiimides, an appropriate alcohol seriesor ultraviolet light.

The GAG substrate should be exposed to the crosslinking agent for aperiod of time sufficient to provide enhancing stability of the GAGstructure. For example, when glutaraldehyde is used as the crosslinkingagent, an incubation period of 72 hours was found to provide goodcrosslinking of the GAG structures. Other crosslinking agents will havedifferent desirable incubation times, as will be apparent to one skilledin the art upon reading this disclosure. Following this incubationperiod, excess cross-linking agent is optionally washed from thestructure.

The present invention is suitable for creating structures comprising anyGAG, including those currently known and those that are as yetunidentified. The structures can be formed from a single GAG, such as astructure containing hyaluronic acid, or can comprise two or more GAGs,such as a structure containing hyaluronic acid and heparin. Thestructures preferably are formed by cross linking hyaluronic acid, thechondroitin sulfates (e.g., netirocan and brevican), keratan sulfate,chitin and/or heparin.

Following cross-linking of the outer layer of the GAGs, a layer of apositively charged coating is added to the GAG structure to resurfacethe structure. Suitable moieties include, but are not limited to,polyvinylamines, polyacrylamides, and polyamino acids, such aspolyaspartic acid, polyglutamic acid, and preferably polylysine. Inaddition, other molecules can be used and the structure subjected tocation exchange to add a positive charge to the surface of the GAGstructure. The positively charged molecules are then crosslinked to theouter layer of GAG by the cross-linking agent remaining in the GAGstructure and/or by the inherent crosslinking abilities of the chargedmoiety. Following the cross-linking of the charged molecule to the outerlayer, the structures can be treated to remove the remainingcrosslinker, e.g., by flushing the structure with an appropriate liquid.Alternatively, when the charged moiety has inherent cross-linkingability, the excess cross linking agent in the core of the GAG structurecan be removed prior to resurfacing.

In either case, the crosslinked outer layers of the structure retain thefree GAG within the core of the structure, while allowing the remainingcrosslinking agent (e.g., glutaraldehyde and/or excess charged moiety)to be flushed from the structure. This removes any cross-linker than mayhave a potentially adverse effect in an in vivo atmosphere.

The crosslinked outer GAG layer and the crosslinked resurfacing layerboth serve as a protective coating to the free GAG to prevent dissolvingof the GAG structure upon contact with a liquid, e.g., a body fluid. Thecoating also allow cells access to the GAG, thus providing accessibilityof GAG in a particular region for desired period, from a matter of hoursor days, up to 3-6 months, and even up to a year or years in anappropriate environment. The resurfaced outer layer of charged moleculefurther provides a surface that promotes cellular attachment, thusallowing cells to access the free GAG present in the GAG structure.

The form of the GAG structure can be controlled by the shape of the GAGsubstrate subjected to crosslinking. For example, when strands aredesired, the GAG solution can be produced as a stream, either into aliquid or onto a solid surface for subsequent drying of the GAGsolution. The GAG solution stream can be introduced in any manner thatcan produce an elongated cylindrical shape, such as with a syringe. Thestructures can be as short as 10 microns, and as long as a meter,although generally the structures are between 0.5 and 5 cm, and moreusually between 1.5 to 2.5 cm in length. The diameter of the strands canbe determined by the diameter of the introduced stream, e.g., by aspecific gauge of a needle used to introduce the GAG solution into theliquid. In another example, when spherical structures are desired, theGAG solution can be introduced as drops, with the diameter of the dropdetermining the diameter of the GAG structure. Following production ofthe GAG structures, the GAG structures are optionally dried prior tofurther processing.

In addition to individual GAG strand, the GAG structures may takeanother form, e.g., a sponge, gauze, spray or gel. These forms may bepredetermined by the GAG substrate, or the form may be determined bycoating a pre-existing structure (e.g., a sponge or gauze) with GAG. Inaddition, the GAG substrate may be molded prior to crosslinking, eitherbefore or after the substrate drying stage.

The methods of the invention also allow the formation of a GAG structureon a surface. This allows a surface to have a coating of free GAG withan exterior coating of cross-linked GAG, which is useful for coatingsurfaces such as sutures or bandages. This allows the surfaces to retainthe GAG, since it will not immediately dissolve upon contact with abodily fluid.

In a particular embodiment of the invention, the resurfaced GAGstructures are composed of hyaluronan (HyA). HyA is a repeatingdisaccharide of [GlcNAcβl-4GlcUAβl-3]_(n) that exists in vivo as a highmolecular weight linear polysaccharide. HyA is found in mammalspredominantly in connective tissues, skin, cartilage, and in synovialfluid, and is also the main constituent of the vitreous of the eye. Inconnective tissue, the water of hydration associated with HyA createsspaces between tissues, thus creating an environment conducive to cellmovement and proliferation.

HyA plays a key role in biological phenomena associated with cellmotility including rapid development, regeneration, repair,embryogenesis, embryological development, wound healing, angiogenesis,immune response and tumorigenesis (Toole, Cell Bioi. Extracell. Matrix,Hay (ed), Plenum Press, New York, 1384-1386 (1991); Bertrand et al. Int.J: Cancer 52:1-6 i (1992); Knudson et al, FASEBJ: 7:1233-1241 (1993>>.The HyA structures of the invention can be used to modulate any of theprocesses which involve HyA. For example, increased binding of HyA toone of its receptors, CD44, has been shown to mediate the primaryadhesion (“rolling”) of lymphocytes to vascular endothelial cells underconditions of physiologic shear stress, and this interaction mediatesactivated T cell extravasation into an inflamed site in vivo in mice. H.C. DeGrendele, et al . J: Exp. Med. 183:1119-1130 (1996); H. D.DeGrendele, et al., Science 278:672-675 (1997). H. C. DeGrendele et al.,J: Immunol. 159:2549-2553 (1997).

In another embodiment, the GAG structures are formed from two or moredifferent GAGs, e.g., an HyA-heparinstructure. These heterologous GAGstructures can comprise two or more different GAGs on the structuresurface, within the structure as free GAGs, or both. Such heterologousGAG structures can be used where it would be advantageous to havemultiple GAGs in the structure, e.g., to modulate a biological functioninvolving multiple GAGs. For example, as discussed above, HyA isinvolved in immune responses, and particularly in leukocyte rolling.Heparin is also involved with leukocyte rolling, adhesion, and migration(Salas A et al. Gut 47:88-96 (2000)), and thus a structure having bothHyA and heparin can be especially useful to modulate such processes.

In another embodiment, the GAG structures can be cultured with cells ofinterest (e.g., fibroblasts, keratinocytes, endothelial cells,macrophages and/or epithelial cells) and the cultured GAG structures canbe used either externally (e.g., in a liquid bandage) or internally(e.g., to enhance healing of an vein or artery). Preferably, the cellsthat are incubated with a GAG structure are autologous to the subject tobe treated with the GAG structure. For instance, when a GAG structure isto be used as a liquid bandage for a specific subject, the cells from aninitial cleaning of the wound can be incubated with the GAG structureprior to application of the liquid bandage. The liquid bandagecontaining such autologous cells can speed the healing process byfacilitating the restructuring of the wound situ without prompting animmune response due to foreign cells. In another example, where a woundto be treated is a wound from which it is difficult to obtain viablecells (e.g., a burn), the cells may be taken from another region in theindividual to be treated. The GAG structures can also be incubated withcultured cells or cells obtained from other sources (e.g., anothersubject).

Methods of Treatment Using the Resurfaced GAG Structures of theInvention

The GAG structures of the invention can be used in any environment thatit would be desirable to have a stable composition of a GAG and/or fordelivery of free GAGs. This includes use of the GAGs in internalregions, such as introduction of the GAG devices into the synovial fluidof a joint, and the use of GAGs in an external fashion, such asapplication of these delivery devices to a wound. The followingdescribed methods of treatment are exemplary to illustrate both internaland external uses of the delivery devices of the invention. Otherpotential methods of treatment and uses of the resurfaced GAG structureswill be apparent to one skilled in the art upon reading this disclosure,and the invention is intended to cover such additional treatments anduses.

Internal Introduction of the GAG Devices of the Invention to TreatArthritis

High-molecular-weight HyA produced by the synovium may functionphysiologically to aid preservation of cartilage structure and preventarthritic pain; both the size and concentration of HyA in synovial fluidare diminished in osteoarthritis (OA).

HyA and albumin act together at normal concentrations to conservesynovial fluid in the presence of raised drainage pressures. Hyaluronanhas the greater effect, acting osmotically by way of a concentrationpolarization boundary layer. Attenuation of this effect in arthriticeffusions with low HyA concentrations is one of several factors limitingfluid accumulation and, hence, the size of the effusion. See, e.g.,Scott D et al., Arthritis Rheum 43: 1175-82 (2000).

In addition, certain sulfated GAGs and polysaccharides—includingchondroitin sulfate (CS), dermatan sulfate, and pentosanpolysulfate—stimulate synovial HyA production, apparently owing to ahormone-like effect triggered by the binding of these polymers tomembrane proteins of synovial cells. See, e.g., McCarty M F et al.,Cancer Treat Rev 54:798-802 (2000). The galactosamine-containingsulfated GAGs have a specific stimulatory effect on HyA synthesis in thesynovial membrane. See, e.g., Nishikawa H et al., Arch Biochem Biophys240:146-53 (1985). A high degree of sulfation of the molecules appearedto potentiate the stimulatory effect.

The GAG structures of the present invention can be used to retainsynovial fluid in the joints of an individual. The structures may becomposed of HyA, other GAGs that stimulate HyA production, or acombination of these. The HyA structures can be used to directlyadminister the HyA into a region to conserve the synovial fluid of aparticular region, e.g, by direct administration of a resurfaced HyAstructure into the joint via injection. Structures having GAGs such aschondroitin sulfate can also be administeredd directly into the joint tostimulate HyA synthesis by synovial cells. In addition, each of theseGAG structures can be cultured with cells (e.g., synovial cells) priorto introduction of the GAG structures into the joint.

An effective amount of a GAG structure to be administered to a subjectto obtain a desired physiological effect, e.g., retention or productionof synovial fluid, can be determined by the caregiver in each case onthe basis of factors normally considered by one skilled in the art todetermine appropriate dosages, including the age, sex, and weight of thesubject to be treated, the condition being treated, and the severity ofthe condition being treated.

Internal Administration of GAG Structures for the Maintenance ofInternal Tissues

The administration of certain therapeutics, such as glucocorticoidadministration, can induce atrophy of skin, bone, and other organs,partly by reducing tissue content of GAGs, and in particular byreduction in HyA. For example, glucocorticoids induce a near-totalsuppression of hyaluronan synthase mRNA in dermal fibroblasts and inosteoblasts, a molecular mechanism contributing to effects such as organatrophy. (Zhang W. et al. Biochem J 349:91-97 (2000)). These effects canbe counteracted by administration of GAG structures of the invention ina manner that will allow the delivery of the desired GAG to a particularorgan. This can be accomplished by, for example, oral administration,intravenous administration, parenteral administration, the implantationof a drug delivery device that would deliver the GAG structures to aparticular organ, and/or a patch that can be applied to an organ, e.g.,a patch either coated with the free GAG having an outer layer ofcrosslinked GAG or comprising GAG structures that allow introduction ofthe GAGs into the tissue. The dosage used can be determined based on themethod of administration as well as on the basis of other factorsnormally considered by one skilled in the art, e.g., the age, sex, andweight of the subject to be treated.

Internal Administration of GAG Structures to Prevent Arterial Restenosis

The GAG structures of the present invention can also be used tostimulate growth of endothelial cells to prevent the narrowing ofvascular tubular walls by the proliferation of the endothelial cells onthe area of trauma. For example, arteries which have been subjected toballoon angioplasty can be treated with the GAG structures of theinvention, and in particular with HyA structures before, during or afterthe balloon angioplasty. Methods for such administration are disclosedin, for example, U.S. Pat. No. 6,022,866 issued to Falk et al. on Feb.8, 2000.

External Administration of Resurfaced GAG Structures to Promote WoundHealing

The GAG structures can also be used externally to promote healing ofexternal wounds (e.g., lacerations, surgical incisions, ulcers, ocularlesions, and burns) by providing the GAG structures to the external siteof the wound. The GAG structures as described herein can be administeredto the wound using a variety of different delivery devices.

Solid Wound Dressings

In one embodiment, the GAG structures of the invention are administeredto the wound site via solid substrates comprising and/or coated with GAGstructures. These solid substrates are intended for use as a temporarydressing on the burns, wounds and other lesions. The dressing itselfforms a barrier against bacterial or other contamination, and theinherent antimicrobial properties of the HA provide a chemical means formaintaining sterility. The dressing preferably remains flexible andfacilitates movement. The GAG structures may be impregnated into thedressing, or may be coated on the dressing, with the coating on the sideto lie adjacent to the patient. Types of wound care dressingsencompassed by the invention include, but are not limited to, alginates,composites, exudate absorbers, foams, gauzes, hydrocolloids, andhydrogels. Exemplary bandages for use with the present inventioninclude, but are not limited to, those described in, for example, U.S.Pat. Nos. 5,718,674, 5,692,937, 5,499,966, 5,376,067, 4,867,821,4,672,956, 4,655,202 and 4,377,159. For a review, see e.g., Bulpitt Pand Aeschlimann D. J Biomed Mater Res (1999) 47:152-169.

The dressing of the invention may be formed from any material known inthe art, including biologically derived materials and syntheticmaterials. Generally, the flexible solid substrate is a syntheticmaterial, and more preferably a woven synthetic material in the form ofa mesh. In an exemplary embodiment, the flexible substrate is amultifilament or monofilament polyester mesh sheet. In another example,a sponge or other substrate may replace the mesh netting, wheremedically appropriate and if its properties match the desired end. Thecoated free GAGs are applied to the substrate of the dressing, e.g., afibrous mesh netting, as an aqueous solution and dehydrated.

The dressing may be used directly, or may be adhered to a backing, e.g.,a self-adhesive backing. Such backing is generally of a flexiblematerial, and usually has an adhesive on the backing surrounding thedressing to allow self-adhesion of the bandage. In one example, thebacking is a flexible strip having a coating of adhesive deposited on atleast the lower planar surface of the strip. A dressing pad of theinvention is attached to, for example, the lower planar surface of thestrip and centered such that a portion of the adhesive strip extendsfrom each end of the wound pad. The wound pad and strip are die cut in apredetermined shape, thereby separating the wound pad and strip into anouter surrounding frame and inner bandage. Exemplary bandages aredescribed in, for example, U.S. Pat. Nos. 5,792,092 and 5,685,833, whichare incorporated herein by reference.

The dressing of the invention can also be separate and held in place bythe elastic forces of a bandage, e.g., a gauze coated with coated freeGAGs held in place by an elastic bandage. Elastic bandages for use inthe invention preferably have good elastic properties, which can beuniform over the width of the bandage. The fabric may be woven orpreferably non-woven. The use of a non-woven fabric in elastic bandagesof the invention can provide a desirable textile ‘feel’ to the surfaceof the bandage. Additionally use of an absorbent non-woven fabric canprovide the bandage with a degree of absorbency for water and bodyfluids such as blood. In one example, an elastic bandage can be usedwhich comprises an inner layer of fabric and an outer layer of fabricbonded to a central layer, such as is described in, for example, U.S.Pat. No. 4,414,970.

A vapor permeable film of plastic material may additionally be joined toone side of the impregnated mesh netting to form an external surface ofthe dressing. The cast dressing is then cut to the desired size ofindividual dressings.

The GAG delivery compounds of the invention may be added in an amountthat allows effective dissemination of the GAG activity from theadhesive preparation. In one embodiment, the GAG structures are coatedonto the bandage using a solution containing from about 0.5% to about20% of the resurfaced GAG structures.

Liquid Bandages

In yet another embodiment of the invention, the GAG structures can beadministered to a wound in the form of a liquid bandage. By “liquidbandage” is meant a flowable substance that can be administered onto orinto a wound that aids in the closure and/or healing of the wound.Liquid bandages can include chemical or biological wound sealants,structural elements that serve as scaffolding for the reconstruction ofthe epithelium, compounds that prevent infection or alleviate pain, anthe like. Liquid bandages of the present invention comprise GAGstructures, and in particular GAG structures composed of HyA. In aparticular embodiment, the GAG structures are cultured with a subject'sautologous cells (e.g., fibroblasts) before administration.

One example of a wound sealant is fibrin sealant, which is comprised offibrinogen and a fibrinogen activator such as thrombin and batroxobin.The fibrinogen activator can be present in various concentrationsdepending on the desired time to form a clot. When the fibrinogenactivator is thrombin, at thrombin concentrations greater than 100 unitsper ml or so in the wound sealant, the fibrinogen concentration becomesthe rate limiting step in coagulation. At concentrations lower thanabout 100μ/ml, the thrombin level is the rate controlling substance inthe wound sealant. Thus, thrombin concentration can be used to controlthe time to gelation.

Another example of a wound sealant is a platelet glue wound sealantcomprising a plasma-buffy coat concentrate as described in, for example,U.S. Pat. No. 5, 733,545. This sealant contains platelets, fibrinogen,and a fibrinogen activator in a concentration sufficient to initiateclot formation.

The GAG structures of the invention may be added in an amount toeffectively treat and/or prevent infection in a wound. Generally, theGAG structures are used in the liquid bandage in a concentration of fromabout 0.5 to 40%, usually from about 1.0 to 20%, more usually betweenfrom about 5 to 10%.

Liquid bandages can be used alone or with additional help from otherclosing devices or methods. For example, liquid bandages can be used inconjunction with sutures, adhesive tape, bandages, and the like toimprove wound closure integrity. Liquid bandages can also be used alone,e.g., in situations involving coagulopathy, friable tissues, adhesionsthat cause bleeding when sutures would be ineffective to control thebleeding, and the like. Other potential uses of liquid bandages of theinvention include sealing vascular suture lines, reinforcing pulmonaryand esophageal staple lines and fixing split-thickness skin grafts. See,e.g., Spotnitz et at., Wound Heating, 77:651-669 (1997).

Sutures

Sutures are often used in the closing of a wound, and currently suturingis the method of choice for closing most surgical wounds. The type ofsuture used will vary depending on the type and extent of the wound, thetissue involved, and a particular patient's healing ability. The GAGstructures of the invention may be used to coat the sutures in an amountthat allows effective activity from the coating. Generally, theconcentration of GAG structure in solution is used in a concentration offrom about 0. 5 to 40%, generally from about 1.0 to 20%, usually betweenabout 5% to 10%. The sutures may also be directly coated with GAG, withfree GAG surrounding the suture and a crosslinked GAG layer surroundingthe free GAG. The sutures are preferably coated with a resurfacingmolecule.

Sutures within the scope of this invention can be of any type used orcontemplated for use in wound closure. The suture can be synthetic ornatural, absorbable or nonabsorbable, or a monofilament or multifilamentin a braided, twisted or covered form. In addition, the sutures can beattached to one or more needles, if desired. Examples of absorbablemonofilament sutures include natural sutures such as surgical gut andcollagen, and synthetic sutures such as homopolymers and copolymers ofp-dioxanone. Examples of absorbable multifilament sutures includesutures prepared from fiber-forming polymers of one or more lactones,e.g., Vicryl.RTM. poly(lactide-co-glycolide) multifilament suture.Examples of non absorbable monofilament and multifilament suturesinclude nylon, polypropylene, steel, polyvinylidene fluoride, linen,cotton, silk, and polyesters such as polyethylene terephthalate (PET).In one embodiment the sutures are nonabsorbable, multifilament sutures,generally polyester sutures, e.g., PET.

Adhesives

The present invention also includes an adhesive compound whichincorporates an adhesive component comprising a GAG structure. The GAGstructures can be homogeneously dispersed throughout the adhesive layer.Active GAG structures of the present composition disassociate from thesurface or allow the GAG activity to be administered over time,delivering healing activity at a distance from the adhesive surface.

The adhesive of the present invention is specifically suited for use inskin contact applications during and after medical procedures, forexample, as an adhesive in surgical drapes, wound dressings and tapes. Aparticular adhesive composition incorporates acrylic polymers and addedtackifiers to form an adhesive which is particularly suited for use inmedical procedures.

An exemplary combination of acrylic polymers to form the adhesivecomposition includes the combination of a low molecular weight solidacrylic polymer and a medium molecular weight solid acrylic polymer in aratio of about 1 to 4, respectively, to optimize the adhesion of theadhesive to skin, cohesion and resistance to cold flow. A low molecularacrylic polymer is a polymer having a molecular weight ranging fromabout 90,000 to about 120,000, while a medium molecular weight acrylicpolymer has a molecular weight ranging from about 140,000 to about160,000. Suitable low molecular weight solid acrylic polymers and mediummolecular weight solid acrylic polymers are available from SchenectadyInternational, Inc. under Product Nos. HRJ-4326 and HRJ-10127,respectively.

The adhesive component of the composition can also include tackifiers asare well known in the art. Tackifiers contemplated include SYLVATEC,ZONAREZ and FORAL which are available from Arizona Chemical andHercules, Inc.

The coated free GAGs of the invention may be added in an amount thatallows effective dissemination of the activity from the adhesivepreparation. Generally, the coated free GAG is used in a concentrationof generally from about 0.5 to 40%, usually from between about 1.0 to20%, more usually from between about 5 to 10%.

Skin Substitutes

Another embodiment of the invention provides skin substitutes thatcoated with free GAG having an outer layer of cross-linked GAG and/orcomprise coated free GAGs. Skin substitutes are commonly used asdressings, especially for burn victims. They can be used to maintain aclean wound environment until skin grafting can be achieved, or may be adressing placed on a partial-thickness wound. The GAG structures caneither be bound to the surface designed to be adjacent to the patient,or interspersed throughout the skin substitute. For a review of suchskin substitutes, see, e.g., Staley et al., Adv. Wound Care 10:39-44(1997).

In one embodiment, the skin substitutes of the invention arebiosynthetic dressings. These include: Biobrane, a flexible nylon fabricimpregnated with collagen and bonded to a silicone membrane; collagenderivatives such as SkinTemp™, Medifil™, Kollagen™, which are typicallyformed from animal collagen; EZ-Derm™, a pigskin impregnated with thepreservative aldehyde; and alginates, which are derived from seaweed andrelease calcium ions to help with homeostasis. These dressings may becoated with the GAG structures of the invention and/or have the GAGstructures impregnated into the fiber of the dressing.

Cultured skin substitutes can also be used in the present invention.These include, but are not necessarily limited to: cultured epidermalautografts, which are produced from a patient's own keratinocytes;Dermagraft, having a collagen base with human neonatal fibroblastsinjected into the matrix; Composite skin, a bilayered cultured skincontaining human fibroblasts on a bovine collagen lattice; Alloderm™, anallograft dermis with all immune cells removed; and Integra™, a bovinecollagen dermis with an outer silicone membrane layer. These skinsubstitutes can be coated with GAG structures of the present inventionto help promote healing of the area treated with the skin substitute.Other similar skin substitutes can also be used, as will be apparent toone skilled in the art upon reading this disclosure. The skin substitutemay be impregnated with the GAG structures, or it may be coated on theside that will contact the patient.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Production of Hyaluronan Strands

First, to prepare the HyA solution for the cross-linking reaction, thecommercially available compound sodium hyaluronate (MW 1.6×106 Daltons;Lifecore Biomedical Inc.) was subjected to a cation exchange reaction.Samples of 0.15 g HyA were put into autoclaved dialysis tubing anddialyzed against cation exchange cellulose (Dowex AG 50W-X4, fromBIO-RAD) at 19/100 ml of deionized distilled water and stirred by amagnetic bar at 4° C. for 2 days. The samples were rinsed with sterilewater, and liquefied HyA was transferred into DMSO. After 2 days offurther stirring, HyA was aspirated into 10-ml syringes.

The HyA solution in the 10 ml syringes was then used to prepare strandsof HyA. The prepared HyA was pushed out of the 10-ml syringes through a25-gauge (0.5 mm) needle to form strands in a beaker filled with 100%alcohol with 10% dimethyl sulfoxide (DMSO) and stored at 4° C.overnight. The strands were placed on 5-mm filter paper and air dried.Then samples in the experimental group were immersed in biological gradeglutaraldehyde (TED PELLA, Inc.) at concentrations of 0.5, 5, 25, and50% aqueous solution for 1, 2, 4, 6, 8, 12, 24, 48, or 72 h at 4° C.After extensive washing in distilled water, the strands were dialyzed inPBS for 48 h in order to reduce residual glutaraldehyde. The samplesthen were stored in PBS overnight.

Fabricated HyA strands crosslinked by 50% glutaraldehyde for 72 hourswere water insoluble and very stable compared to the groups crosslinkedwith lower concentrations of glutaraldehyde or for shorter incubationtimes (Table 1). HyA strands, crosslinked for 72 hours or longer with50% glutaraldehyde maintained their shape for at least 5 days to over 3months (FIG. 1, 2), while the groups crosslinked by 50% glutaraldehydefor 48 hours would dissolve in PBS or distilled water after 2 days. Whenlower concentrations of glutaraldehyde were used, such as 25%glutaraldehyde, the strands dissolved in PBS in from 5 min to 12 hourswhile all other experimental groups crosslinked with concentrations ofglutaraldehyde less than 25% immediately dissolved in PBS or distilledwater.

Example 2 Resurfacing Crosslinked HyA Strands

The crosslinked HyA strands were then resurfaced to determine theability of different surface to adhere to cells. Solutions of 1%L-glutamine (Sigma), 1% glycine (amino-acetic acid, BIO-RAD), 50 mg/1 mLof poly-L-lysine (Sigma), and 10 mg/ml of poly-D-lysine (Sigma) werefreshly prepared. Sets of HyA strands crosslinked with 50%glutaraldehyde were immersed in one of four solutions for 1 hour, withan additional set receiving no resurfacing treatment. After immersion,the strands were washed thoroughly with distilled water.

The crosslinked, resurfaced HyA strands were then prepared forvisualization of the external coating of the strands. The crosslinked,resurfaced HyA were impregnated in the tissue culture medium. Thecultured HyA strands were then washed in PBS for 30 min, fixed in 10%neutral formaldehyde for 1 hour, and washed again in PBS for 30 minprior to staining. Strands were then incubated with Alcian Blue solutionfor 30 min, rinsed with PBS three times for five min, followed byrinsing with distilled water 3 times for 3 min. The strands were put ona glass slide and mounted with Gelatin. Photographs of intact, stainedHyA strands at 400× magnification showed that the resurfaced crosslinkedHyA strands have a distinct structure, with a light green-blue coloroutlayer of the crosslinked HyA strands.

The stained crosslinked HyA strands were then put through a series ofalcohol baths increasing in concentration from 70% to 100% alcohol. Thestrands were kept in 100% alcohol for 0.5 hours, changing the alcoholthree times, to make the surface of the strands tough and rigid. Thenthe surface of the strands were cracked with a glass cover slip toreveal the internal core of the strands. The stained outer layersdisplay crosslinking only at the periphery of the strand, with aninternal core of unstained, free HyA (FIGS. 4A-4C).

Example 3 Fibroblast Growth on Resurfaced HyA Strands

Each of the 4 different sets of resurfaced HyA strands were tested fortheir ability to provide bioactive HyA to cells. Monolayers of ratfibroblasts in their growth phase, approximately 3 days after splitting,were trypsinized for 15 min to form a suspension. Cells were pooled bycentrifugation at 2000 rpm for 10 min and resuspended in DMEM,4-6×10⁵/cm². A syringe with a cut-tip needle was used to aspirate andpush out the cellular medium three to five times to homogenize theclumps of cells. Following homogenization, the fibroblasts were gentlyapplied to the surface of the HyA strands in 35×10-mm culture dishes. 1ml of medium (DMEM+10% fetal bovine serum) was added to each of theculture dishes. The cultures of cell-seeded HyA strands were incubatedat 37° C. and observed at 24 h intervals for 7 days using an invertedmicroscope. The culture dishes were then washed in PBS for 30 min, fixedin 10% neutral formaldehyde for 1 hour, and washed again in PBS for 30min prior to staining (FIGS. 3A-3D).

Specimens were incubated in 0.3% H₂O₂ for 30 min, washed in PBS, andblocked with normal horse serum for 30 min. Following blocking, thespecimens were incubated overnight with PCNA antibody (mouseanti-proliferative cell nuclear antigen, pc-10, DAKO) at 1:200 dilution.Afterwards specimens were treated with the avidin-biotin-complex kit(Vector), developed with DAB tablets (Sigma), and mounted with gelatin.Other specimens routinely were stained with H&E or Alcian Blue. A Baxtercell counter was used to count cells attached to a 1-cm segment of a HyAstrand. Proliferating cells were marked by PCNA-positive staining. Theproportion of positively stained cells was assessed and graded on ascale of 0 to ++++ (0=negative immunoreactivity; +=1-25 percent;++=26-50 percent; +++=51-75 percent, and ++++=76-100 percent).

Although 50% Glut-crosslinked HyA strands dimensionally were stable inculture medium, there was no attachment of fibroblasts followinginoculation with the non-resurfaced HyA strands. After resurfacing withpolylysines, the adhesive ability of the HyA strand surface wasenhanced, especially with poly-D-lysine, whereas L-glutamine and glycinecoating did not facilitate cell attachment. Poly-D-lysine improvedadhesion most effectively, attaching 50-100 cells per centimeter of HyAstrand, and poly-L-lysine was effective to a lesser extent, attaching40-80 cells per centimeter. The results are summarized in Table 1: TABLE1 Adhesion and Proliferation of Fibroblasts on HyA Strands ResurfacedWith One of Four Different Amino Acids or Polypeptides Resurfacing CellAttachment Count Proliferation Treatment (Average # per 1 mm HyA Strand)Proliferation None 0-2 − Poly-D-lysine  50-100* ++ Poly-L-lysine  40-80*++ Glycine 0-7 + L-glutamine 0-5 +*p < 0.01.

Also, both polylysines allowed cell growth on the surface of the HyAstrands for at least 1 month. Fibroblasts on the strands stainedpositively for PCNA but not as strongly as for those that grew besidethe strands on the bottom of the culture dish. Cells adhering to thepolylysine-coated strands showed more proliferation (26-50% PCNAimmunostaining) than cells on the other treatment. PCNA mainly waslocalized to cell nuclei, but some staining also was scattered in thecytoplasm.

Biocompatibility of the resurfaced glutaraldehyde-crosslinked withfibroblasts was demonstrated using the cell-seeded HyA strands. Thefibroblasts grew in vitro without inhibition or toxicity along and overthe HyA material as well as on the dish bottom. Visualization of theinteraction of the cells with the HyA strands was achieved using theAlcian Blue staining protocol as described in Example 2. The fibroblastson the HyA strand have been shown to extend into the core region of thestrand, where the free HyA is contained, and therefore allows the cellsaccess to the free HyA (FIGS. 4A-C).

Example 4 Inoculation with HyA Strands in Vivo

Each of the 4 different sets of resurfaced HyA strands were also testedfor their ability to provide bioactive HyA following in vivoimplantation. Five Wistar male rats weighing about 400 g wereanesthetized with intraperitoneal injections of 4% chioral hydrate. Foursegments (of 2 cm each) of the fibroblast-seeded HyA strands wereimplanted in a subcutaneous pocket in the right chest of the rat, andthe incision was closed with a continuous 4.0 nylon suture.

After 2 or 4 weeks the rats were sacrificed, the implants withsurrounding tissue were removed, and the specimens were fixed inbuffered formaldehyde for 2 hours. The specimens were processed,paraffin embedded, and cut into 10 mm sections onto slides. The slidesthen were deparaffinized and rehydrated in preparation for staining.

In vivo, implantation for 4 weeks in rats did not elicit any apparentinflammatory or necrotic response. Under microscopic examination, HyAstrands maintained their shape and cells grew along the strands. PCNAstaining of implanted strands showed activity proliferating cells,graded at “++”, on strands resurfaced with poly-L-lysine andpoly-D-lysine. Surrounding connective tissue infiltrated the spacearound the implant, but few inflammatory cells, macrophages, or lymphoidcells migrated to the area.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Example 5 Wound Treatment with HyA Strands

Thirty male or female SD rats weighing 400 grams were fullyanesthetized. Two parallel full-thickness incisions 1.5 cm in lengthwere made 2 cm apart from each other on the dorsal skin of their upperback. HA strands with or without cultured cells, both 6 cm long, wereimmediately inserted into the acute full-thickness bed in one of twowounds on each rat. The contralateral wounds received only saline washbut otherwise were processed identically, without suturing toapproximate wound edges.

Five rats were sacrificed at the time points: 1 day, 3 days, 7 days, 2weeks, 4 weeks and 8 weeks post-surgery. The wound regions with thesurrounding tissues were excised and fixed in 10% buffered formaldehydeand routinely processed, paraffin embedded, and cut into 5 micronsections perpendicular to the skin surface and transverse to the wounds.Half the tissue slides were stained with routine hematoxylin and eosin(H&E) and Alcian Blue. Immunohistological staining and the quantitativeevaluation of the HA and HA+cell graft were performed as above describedin EXAMPLE 3.

The wound closure time required for the experimental and control groupsare shown in TABLE 2. Wounds treated with HA grafts or HA+cell graftsclosed in 15.85±4.77 and 16.15±4.66 hours respectively. There was nosignificant difference between these two treatment groups (p>0.5). Incontrast to the HA grafts, the controls had an average of 114.2±18.41hours for the final closure of the wound which is significantlydifferent (P<0.01) compared to the experimental wounds. About half ofthe control wounds were still bleeding or had fresh clots in the centerof the wounds after three days.

TABLE 3 demonstrates the comparative scar areas among the three woundtreatments under microscopy. Because all tissue specimens were takenvertically from the centers of the linear incision wounds, the measuredareas were as uniform as possible. The averages of the cross-sectionalwound areas were: HA only graft 0.151 mm²±0.035, HA+cell 0.143 mm²±0.036and controls 14.434 mm²±1.175. There was less fibroblast proliferation,and almost no inflammatory cell infiltration in the experimental skintissues compared to the tissue from the control group.

FIGS. 5 (A-D) illustrates gross incision wound healing after HA graftswere placed into wounds. There was a dramatic difference in the closurerate between HA graft wounds and control wounds. Most HA only or H+celltreated wounds closed rapidly in a fine line without contracture. Someshowed even faster closure: one hour after the graft was implanted,bleeding stopped and the wounds were closed. Afterwards, there was nobleeding when the rats moved. Microscopically, cutaneous appendages(including hair follicles, sweat glands and sebaceous glands) were foundin the corresponding healing area. Compared to the experimental wounds,contralateral control wounds required longer time to achieve closure.These wounds demonstrated the routine course of non-sutured woundcontracture, being contracted into bumps. Microscopically, the largerscar area of the controls contained more eosin stained disorganizedmatrix (collagen fiber bundles) with increased fibroblast andinflammatory cell numbers and increased vascularity. Fewer skinappendages could be seen in the healing area (FIGS. 6A and 6B).

1. A glycosaminoglycan structure, comprising: a core of freeglycosaminoglycan; a layer of crosslinked glycosaminoglycan surroundingsaid core; and a layer of a charged molecule surrounding saidcrosslinked glycosaminoglycan; wherein the structure is stable both invitro and in vivo, and wherein the structure effectively binds to cells.2. The glycosaminoglycan structure of claim 1, wherein the structurecomprises a single glycosaminoglycan.
 3. The glycosaminoglycan structureof claim 1, wherein the structure comprises at least two differentglycosaminoglycans.
 4. The glycosaminoglycan structure of claim 1,wherein the structure comprises hyaluronan.
 5. The glycosaminoglycanstructure of claim 1, wherein the charged molecule is a positivelycharged polyamino acid.
 6. The glycosaminoglycan structure of claim 5,wherein the charged molecule is polylysine.
 7. The glycosaminoglycanstructure of claim 1, wherein the structure is a strand of about 0.5 toabout 5 cm in length.
 8. The glycosaminoglycan structure of claim 1,wherein the structure is spherical.
 9. A composition for introducing aglycosaminoglycan to a subject, said composition comprising: aglycosaminoglycan structure, wherein said glycosaminoglycan structurecomprises a core of free glycosaminoglycan, a layer of crosslinkedglycosaminoglycan surrounding said core; a charged molecule surroundingsaid crosslinked glycosaminoglycan layer; and an excipient.
 10. Thecomposition of claim 9, wherein the composition further comprisescompounds that promote wound healing.
 11. The composition of claim 9,wherein the composition further comprises cells adhered to saidglycosaminoglycan structure, wherein said cells are characterized by anability to enhance wound healing.
 12. The composition of claim 11,wherein the cells are from the subject to be treated.
 13. A method forproducing a composition for introducing a glycosaminoglycan to asubject, comprising the steps of: exposing a glycosaminoglycan substrateto a liquid comprising a crosslinking agent, wherein the crosslinkingagent is present in the liquid in a concentration of between 35% and85%; incubating the glycosaminoglycan solution with the liquid for atime sufficient to allow crosslinking of the glycosaminoglycans at theperiphery of the substrate to create a glycosamino-glycan structure; andexposing the glycosaminoglycan structure to a charged molecule to form acoating of the charged molecule surrounding the glycosaminoglycanstructure; wherein the composition is characterized by in vivostructural stability and an ability to adhere to cells in vivo.
 14. Themethod of claim 13, wherein the crosslinking agent is present in theliquid in a concentration of between about 45% and about 75%.
 15. Themethod of claim 13, wherein the crosslinking agent is selected from thegroup consisting of formaldehyde, vinyl sulphone, biscarbodiimides, andcarbodiimides.
 16. The method of claim 13, wherein the crosslinkingagent is glutaraldehyde.
 17. The method of claim 13, wherein the methodfurther comprises the step of removing excess crosslinking agent fromthe glycosaminoglycan structure.
 18. The method of claim 13, furthercomprising the step of preparing a glycosaminoglycan substrate.
 19. Themethod of claim 13, wherein the glycosaminoglycan is selected from thegroup consisting of hyaluronan, chondroitin sulfates, laminin, keratinsulfate, chitin and heparin.
 20. The method of claim 13, furthercomprising the step of forming the glycosaminoglycan substrate.